Ebola virus (EBOV) causes periodic outbreaks of severe viral hemorrhagic fevers in Africa with high mortality rates in infected patients. EBOV is classified as a Category A bioweapons agent by the Centers for Disease Control and Prevention (CDC) because of its highly infectious nature. Currently, there is no FDA approved vaccine or antiviral drug that is effective against EBOV infections. Moreover, rapid progression of EBOV infection will offer little opportunity for developing acquired immunity in an infected population. Therefore, there is a critical need for development of effective antivirals to respond to EBOV outbreak or bioterrorist attack. EBOV infection is initiated by the fusion between viral and host cell membranes, which is mediated by the viral envelope glycoprotein (GP). This selective interaction between EBOV-GP and host cell surface receptor molecules is essential for the initiation and establishment of the infection. Blocking of EBOV entry will lead to suppression of viral infectivity early in its life cycle. Our goal is to develop small molecule inhibitors of EBOV infection that can be used either prophylactically to a potentially EBOV exposed population or therapeutically during the post-infection period. In preliminary studies of this project, we have identified eight EBOV entry inhibitors of different chemotypes, which are active against infectious EBOV (IC50 = 20 ?M) and have low selectivity indices. The potential inhibitors and their derivatives will be further optimized and characterized by medicinal chemistry to increase their anti-EBOV potency (IC50 <1 ?M), and selectivity indices (SI>10). In addition, we will continue to screen a large number of molecules in a search for inhibitors with higher selectivity to provide new entry points for chemical optimization. The research team represents a collaborative effort by four experienced research and drug discovery/development groups; 1)Microbiotix, Inc. will conduct the medicinal chemistry, initial biological assays, mechanism of action studies and in vitro ADME evaluation; 2) Southwest Foundation for Biomedical Research (SFBR) will conduct the in vitro and in vivo EBOV infection evaluation; 3) Univ. of Illinois at Chicago (UIC) will conduct the mechanism of action studies; and 4) the Harvard Medical School will conduct the EBOV-GP-receptor binding studies. The major milestone of this proposal is to select 2-3 EBOV entry inhibitor candidates that will be advanced to Investigational New Drug (IND) enabling toxicology and safety pharmacology studies. Therefore the specific aims of this application are to: 1. Screen compound libraries to identify selective noncytotoxic inhibitors of EBOV entry; 2. Optimize the potency and selectivity of hit compounds and validate their antiviral activities; 3. Determine the mechanism of action (MOA) of the initial hit series and prioritize screening hits; 4.Identify EBOV inhibitors with in vitro ADME properties suitable for i.v. and oral dosing; and 5. Evaluate EBOV inhibitors in a guinea pig model of EBOV infection.